Published: 13 August 2001 Received: 9 July 2001

Accepted: 13 August 2001BMC Genetics 2001, 2:13This article is available from: http://www.biomedcentral.com/1471-2156/2/13 2001 Maca-Meyer et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any non-commercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact info@biomedcentral.com

Abstract Background: The phylogeographic distribution of human mitochondrial DNA variations allows a genetic approach to the study of modern Homo sapiens dispersals throughout the world from a female perspective. As a new contribution to this study we have phylogenetically analysed complete mitochondrial DNA(mtDNA) sequences from 42 human lineages, representing major clades with known geographic assignation. Results: We show the relative relationships among the 42 lineages and present more accurate temporal calibrations than have been previously possible to give new perspectives as how modern humans spread in the Old World. Conclusions: The first detectable expansion occurred around 59,00069,000 years ago from Africa, independently colonizing western Asia and India and, following this southern route, swiftly reaching east Asia. Within Africa, this expansion did not replace but mixed with older lineages detectable today only in Africa. Around 39,00052,000 years ago, the western Asian branch spread radially, bringing Caucasians to North Africa and Europe, also reaching India, and expanding to north and east Asia. More recent migrations have entangled but not completely erased these primitive footprints of modern human expansions.

Figure 1Phylogenetic network based on complete mtDNA genome sequences. Nomenclature of individuals is as in Table 1. Numbers along the links refer to nucleotide posi-tions; suffixes are transversions; underlining indicates recurrent mutations; the order of the mutations on a path not interrupted by any branching or distinguishednodes is arbitrary. The same topology was supported by bootstraps, using NJ and 1000 replicates; the bootstrap values higher than 50% are shown over the branches.The star shows the position where the chimpanzee sequence roots in the network. http://www.biomedcentral.com/1471-2156/2/13BMC Genetics 2001, 2:13 http://www.biomedcentral.com/1471-2156/2/13

crucial aspects of human history, such as the probable however, parallel mutations should be avoided in theirorigin and approximate dating of migrations into the global affiliations. As can be expected from haplotypes ofNew World [3] and Polynesia [4,5], and quantitative es- well-differentiated haplogroups the majority of muta-timations of the relative Paleolithic and Neolithic contri- tions are in the external branches of the tree, includingbutions to the extant European mtDNA diversity [2]. At those that specifically define them [2]. Nevertheless, it isthe other end of the phylogenetic tree, the ultimate coa- well known that in population studies these main lineag-lescence of all worldwide mtDNA lineages into Africa has es sprout into several sub-clusters sometimes with inter-favored, since the beginning, the recent African origin esting geographic localization. In the cases wherehypothesis for all modern humans [6]. The analyses of representatives of these sub-clusters have also been ana-the complete mtDNA sequence of 53 humans of diverse lyzed, it is evident that the African ones are at the sameorigins [7] have added statistical support to this hypoth- level of divergence as non-African clusters. More infor-esis. However, as the current definition of the major hap- mation of cluster structure in Africa is necessary. In nonlogroups is not based on total genomic sequences, there African groups, two haplotypes belonging to sub-haplo-is not yet a clear resolution of their basal relationships. group U2 have a divergence similar to that found be-This genomic phylogenetic reconstruction is necessary to tween other sub-clusters of the Caucasian U haplogroup.infer the early human dispersal routes after the African One of them, lacking mutations 16129C and 15907, thatexodus. We present the phylogenetic network of 42 com- are present in all western Eurasian representatives, re-plete mtDNA sequences including representatives of the sembles haplotypes found in India [9]. The proposed in-major haplogroups. Based on their relative clustering clusion of haplogroup K into the U cluster [10] isand coalescence ages we propose a tentative model of the confirmed, being U7 its most probable related sub-clade.way the Old World could have been colonized by modern Main Asian haplogroups belong to two different majorhumans. clusters, whereas A and B rooted with Caucasoid haplo- groups, C, D, G and M constitute a monophyletic cluster.Results and Discussion Likewise, African haplogroup L3 is more related to Eur-The phylogenetic network of the 42 mtDNA sequences asian haplogroups than to the most divergent African(Fig. 1) was free of reticulations when mutations [8] 150, clusters L1 and L2. Chimpanzee rooting shows that the152, 303i and 16519 were omitted in its construction. The oldest lineage of extant modern humans is the Africantree topology was the same as the bootstrap supporting L1a cluster. In addition, the significant bootstrap valuesneighbor joining tree. We detected 35 parallel substitu- on the deep African branches reinforce the statisticaltions from 124 variable positions (28%) in the non-cod- support that the out of Africa hypothesis has obtaineding region (1,122 bp in length), and 45 from 409 (11%) in through a parallel genomic mtDNA study [7]. We havethe coding region (15,447 bp in length). Shared muta- estimated a minimum total coalescence for modern hu-tions in basal branches of the tree relate haplogroups, man lineages from 156,000 to 169,000 years before present (yr BP). The two subsequent ancient splits also happened inside Africa, originating the L1b/c and L2 haplogroups with ages of 122,000132,000 yr BP and 85,00095,000 yr BP respectively. These three clades still have an overwhelming sub-Saharan African implan- U5 A tation. The next branching (Fig. 2), dated between 39.000-53.000 B 59,00069,000 yr BP, also occurred in Africa but com- U6 U2 C D prising clades currently found only in this continent L3 M G (L3), and others with a first expansion out of Africa. To- 59.000-69.000 day, L3 derivatives are present in nearly all the African L2 L1b/c populations. This ancient spread inside Africa has been L1a directly detected by the ages of several sub-clade expan- sions [11] and indirectly confirmed by genetic admixture, involving archaic and modern autosomal gene alleles, detected only in Africa [12]. The coexistence in African populations of very divergent non-recombining lineages may erroneously bias demographic estimations based onFigure 2Geographic dispersal routes and minimal estimated ages of pair-wise nucleotide differences [11]. Two hypotheticalmajor human expansions in the Old World, deduced from routes for the Asian colonization have been proposedthe age and geographic localisation of main mtDNA haplo- [13], one through Central Asia and one through Southgroups. Asia. Coincidentally, we detect at least two independent lineages spreading out of Africa. One comprises all M de-BMC Genetics 2001, 2:13 http://www.biomedcentral.com/1471-2156/2/13

a 1, This work; 2, GenBank accession number X93334; 3, H and I references [34], we have added for the comparisons the 263, 311i and 16519 mutations in both sequences and 00073 in the I sequence; 4, revised Cambridge reference, GenBank accession number NC 001807; 5, Positive con- trol [35], for comparisons we added 1438; 6, MELAS, P-1 (G) and FICM (D) [36]; 7, (ref [37]); 8, GenBank accession number D38112, for compar- isons we added 311i.

rivatives that radiated 30,00057,600 yr BP. Subse- like radiation of these clades suggests that this wide geo-quent expansions of this clade have been found in India graphic colonization could have happened in a relatively[9] and Eastern Asia where it possibly originated and ex- short time. Genetic support for this southern spread of Mpanded as haplogroups C, D, G and others [14]. The star- through Ethiopia and the Arabian Peninsula along SouthBMC Genetics 2001, 2:13 http://www.biomedcentral.com/1471-2156/2/13

Asia has been recently proposed due to the presence of northern India [9] but is present in Mongolia [26], fa-subclade M1 in Eastern Africa [15]. However, a posterior voring a Central Asian route for the expansion of thesereturn from Asia to Africa of these lineages is a more prominent Asian haplogroups. Two additional cladesplausible explanation because the genetic diversity of M join haplogroups J and T and haplogroups H, V and HVis much greater in India [9] than in Ethiopia [15]. In fact, respectively. Derivatives of at least some of them areM1 could be a branch of the Indian cluster M as ancestral found in Europe, North Africa, Central Asia and even In-motifs of the African M1 are found in M*, M3 and M4 In- dia, but the most probable origin for all these expansionsdian subclusters [16]. Furthermore, one of the most de- is the Near East-Caucasus area [2,17,27]. Finally, clusterrived M3 haplotypes in India (10398, 10400, 16086, U seems to have suffered a radial spread (Fig. 2), giving16129, 16223, 16249, 16259, 16311) has all the basic sub- subsequent diversification in different geographic areas.stitutions that defined the Ethiopian clade, excepting the Three sub-haplogroups, U2, U5 and U6 had their majorhighly variable 16189 [9]. This supposed Indian expan- expansions in India, Europe and North Africa respective-sion to the west also reached northern areas since ly. U2 split in two branches, one, characterized by muta-evolved representatives of M4 have been also detected in tions 16129C and 15907, is geographically scattered fromCentral Asia [17]. We may consider the upper bound for Western Europe to Mongolia [2,26] but has not been de-this return to Africa 25,00047,000 yr BP, the age calcu- tected in North Africa. The other reached India where itlated for M1 in Eastern Africa based on HVSI sequences gave origin to several sub-clusters with global frequen-or 33,00063,000 obtained using RFLPs [15]. cies around 10% being, after its predecessor haplogroup M (53%), the second most abundant haplogroup in IndiaThe other major branch that left Africa gave rise mainly [9]. U7 with a minor implantation in Europe but third into Caucasoid lineages which is congruent with a northern frequency in India [9] and also not detected in North Af-route through the Levant. With a lower bound of rica might have had a similar expansion as U2. The main43,00053,000 yr BP this branch spread into at least radiation of haplogroup U5 occurred in Europe. It hasthree main clusters. One comprises haplogroups X and A been stated that this lineage entered Europe during thewith only a shared mutation between them and different Upper Paleolithic [2], most probably from the Middlegeographic distributions. Whereas A is widespread in East-Caucasus area. The great divergence found here forAsia, X is mainly restricted to Europe. Curiously, repre- the two U5 representatives is in agreement with the oldsentatives of both clusters have been detected in native age proposed for this haplogroup. Finally, U6 traces theAmericans raising the possibility that some American In- first detectable Paleolithic return to Africa of ancientdian could have European ancestry [18]. Nevertheless, X Caucasoid lineages. It has been mostly found in North-haplotypes have recently been detected in Central Asia. west Africa, with a global estimated age of 47,000 yearsThese Asian X haplotypes lack the 225A mutation, as the [28] reflecting an old human continuity in that rathermajority of the American X, pointing to this area as the isolated area. The fact that in Europe it has only been de-most probable source for the dispersal of the New World tected in the Iberian Peninsula [29] rules out a possiblefounders [19]. The second cluster groups minor haplo- European route, unless a total lineage extinction in allgroups W, I and N1b, the three are present although in the path is invoked. On the other hand, its presence inlow frequencies in Europe, Near East and Caucasus but Northeast Africa [30], albeit in low frequencies, reinforc-only I and N1b have been also detected in Egypt and Ara- es its way through North Africa. A third possibility couldbia [2]. The last group radiated around 39,00052,000 be that this lineage never went out of Africa but its coa-yr BP, giving at least four ancestral clusters. One of them lescence with clades which all had prominent expansionsoriginated haplogroup B that expanded to Eastern Asia, in Eurasia weakens this option. U3 has also been foundreaching Japan and southeastern Pacific Archipelagos with a comparatively higher frequency in Northwest Af-[20,21]. In early studies, this clade was defined by the 9- rica [29] and might have followed the same route as U6,bp COII-tRNALys deletion but after that it has been however, as its star-like expansion in the Caucasus hasfound with independent origins on other haplogroup been dated around 30,000 yr BP [30], it most probablybackgrounds [2224]. In this study we have detected reached Africa in a posterior expansion. This out of Afri-this deletion on an Iberian haplotype belonging to haplo- ca and back again hypothesis has also been suggested forgroup I. Curiously, it was also found in an Italian haplo- Y-chromosome lineages [31]. Subsequent Neolithic andtype I [25]. However, the 9-bp deletion was absent in a historic expansions have doubtlessly reshaped the hu-wide screen that we carried out on Iberian and North- man genetic pool in wide geographic areas but mainly aswest African I haplotypes. The detection in two Mediter- limited gene flow, not admixture, between populations.ranean populations of I haplotypes harboring the 9-bp Consequently, the continental origin of the major haplo-deletion points to the existence in this area of a subset of groups can still be detected and the earliest humanI haplotypes that share a recent common ancestor. As routes inferred through them.happens with A, haplogroup B has not been found inBMC Genetics 2001, 2:13 http://www.biomedcentral.com/1471-2156/2/13

Table 2: Oligonucleotide pairs used in the amplification and sequencing